Field of Invention
The present disclosure provides compositions and methods utilizing double strand ribonucleic acid (dsRNA) to control the insect pest, the brown marmorated stink bug (Halyomorpha halys). More particularly, the present invention relates to several specific synthetic dsRNAs that induce RNA interference (RNAi) in the target insect.
Background
Insect pests comprise a broad collection of animals that adversely affect urban and rural plants and animals. Halyomorpha halys (stål) (Heteroptera: Pentatomidae), commonly known as the brown marmorated stink bug (BMSB), is an invasive insect native to Asia (China, Taiwan, Korea, and Japan) that has emerged in the last decade as an important major insect pest in the United States, Canada and Europe. BMSB is a polyphagous piercing-sucking feeder causing damage by inserting their feeding stylets into the fruit, pods, buds, leaves, and stems of more than three hundred known plant hosts. This pest poses a considerable ecological and economic threat—billions of dollars annually—to specialty crops such as apples, stone and pome fruits, grapes, ornamental plants, vegetables, seed crops, as well as staple crops such as soybean and corn. BMSB has recently been found to be most abundant on extended fruiting plants such as sweet corn, okra, eggplant and bell pepper as these tend to support a longer life cycle (Zobel et al, J. Econ. Entomol., (2016)). BMSB has rapidly expanded its range from the original single point of accidental introduction and establishment in the Allentown, Pa. area in the late 1990s. Damage has been particularly extensive in the U.S. Mid-Atlantic Region (DE, MD, PA, NJ, VA, and WV) and has been detected in 42 states and Canada, as well as Europe (Xu et al, Biol. Invasions, (2014) 16:153-66; Leskey et al, Outlooks on Pest Management, (2012) 23(5):218-26; Hoebeke, Proc. Entomol. Soc. Wash., (2003) 105:225-37). In addition to being an agricultural pest, BMSB is also a nuisance pest because it invades indoor structures such as houses, schools and other indoor spaces that provide a safe hiding area in the fall to overwinter until spring for mating and egg laying (Leskey et al., supra). BMSB may trigger an immunological response in humans by causing allergic reactions leading to conjunctivitis and rhinitis in individuals sensitive to aeroallergens, or through contact dermatitis upon exposure to the crushed animal (Mertz et al, J. Allergy Clin. Immunol., (2012) 130(4):999-10001; Anderson et al, Dermatitis, (2012) 23(4):170-72). As yet, no effective control methods have been discovered, leading us to investigate the possibility of using RNA-mediated interference (RNAi) as an approach to control this insect.
The discovery of RNA-mediated interference (RNAi) has facilitated research to understand gene function and regulation. RNAi is a well described gene regulatory mechanism wherein exogenous dsRNA is introduced into the cells of eukaryotic organisms and targets degradation of host cell mRNAs containing sequences complementary to the dsRNA (Mello and Conte, Nature (2004) 431:338-42). RNAi depletes host mRNA either by transcriptional gene silencing, or at a posttranscriptional level, thereby affecting translation of the protein (Ambros, Nature (2004) 431:350-55). RNAi takes advantage of internal cellular defenses against the presence of dsRNA, which typically indicates an on-going viral infection.
Virally induced dsRNA-activated innate immune responses in mammalian cells initiate a chain of events such as inhibition of protein synthesis, transcriptional induction of antiviral genes, and leading to cell death lead to the discovery of dsRNA mediated RNA interference (RNAi) (Barber, G. N., Cell Death and Differentiation, (2005) 12(6):563-70; Gantier & Williams, Cytokine and Growth Factor Revs., (2007) 18(5-6):363-71). RNAi is a phenomenon of posttranscriptional gene silencing mediated by dsRNA or small interfering RNA siRNA. RNAi involves sequence specific degradation of a target gene mRNA mediated by dsRNA. These dsRNAs are subsequently cleaved to 19-21 bp siRNAs fragments by member of the RNase III superfamily of bidentate nucleases known as dicers (Bernstein et al, Nature, (2001) 409(6818):363-66; Ketting et al, Genes & Develop., (2001) 15(20):2654-59). These short RNAs unwind and together with a multi protein complex, RNA-induced silencing complex (RISC) and acts as template for complementary mRNA recognition. This RISC-RNA complex in conjunction with argonaute multi-domain protein containing an RNAse H like domain are corollary for target degradation hence silencing the gene resulting in translational blockage (Martinez et al, Cell, (2002) 110(5):563-74; Bartel, D. P., Cell, (2004) 116(2):281-297).
RNAi was first discovered in the nematode Caenorhabditis elegans (Fire et al, Nature, (1998) 391(6669):806-11). Since the inception of RNAi as an efficient method of gene silencing, it has been successfully applied in insects for dsRNA-mediated gene regulation. dsRNA delivery system to insects and insect cells included feeding (Baum et al, Nature Biotech., (2007) 25(11):1322-26; Timmons & Fire, Nature, (1998)395(6705):854), soaking (Saleh et al, Nature Cell Biol., (2006) 8(8):793-802), microinjections (Amdam et al, BMC Biotech., (2003) 3:1), and other techniques (reviewed in Huvenne & Smagghe, J. Insect Physiol., (2010) 56(3):227-35) were used for systemic dsRNA uptake. To date, RNAi technology has been successfully applied to hemipteran insects indicating depletion of calreticulin and cathepsin-L (Jaubert-Possamai et al, BMC Biotech., (2007) 7:63), Coo2 (Mutti et al., J. Insect Sci., (2006) 6:1-7), ApAQP1 of insect aquaporins (Shakesby et al, Insect Biochem. Mol. Biol., (2009) 39(1):1-10) and hunchback (Mao & Zeng, PLoS One, (2012) 7(11): e48718) in the pea aphid (Acyrthosiphon pisum) by mechanical microinjection, or through artificial diets. A recent report demonstrated depletion of the catalase gene in BMSB utilizing mechanical microinjection as a mode of dsRNA delivery. But they also raised a concern alongside that mechanical puncture may stress the animals that may induce unexpected variations in the expression of the housekeeping genes affecting validation of RNAi through qRT-PCR (Bansal et al, PLoS One, (2016) 11(5):e0152730).
Reported herein are compositions and methods for controlling The depletion of putative genes varied in diminishing the target mRNA but higher concentrations of dsRNA were required for depletion of certain genes, including HP C37491, AK, Ftz F1 and JH. Vitellogenin, the major egg yolk protein displayed a significant reduction with a gradual increase in dsRNA concentration but was hyperexpressed at the peak concentration. The importance and correlation in the sesquiterpene biosynthesis pathway was also investigated where depletion of JH negatively affected the expression of FPPS1, an enzyme important in synthesis of the male aggregation pheromone. Here we have demonstrated that exogenously synthesized dsRNA can elicit RNAi response in BMSB through an oral delivery method. This efficient vegetable-mediated dsRNA delivery method suggested that this RNAi pathway might be directed for the control of invasive insect pests of agriculture.